It is generally believed that acute pancreatitis is triggered by the intra-acinar cell activation of digestive enzyme zymogens including trypsinogen. However, the mechanisms responsible for intra-acinar cell zymogen activation and the subsequent events which determine the severity of pancreatitis are poorly understood. The proposed studies will build on our previous observations to explore these issues. The Specific Aim #1 studies will examine the relationship between digestive zymogen/lysosomal hydrolase co-localization and zymogen activation. We will determine if co-localization leads to or results from zymogen activation. The Specific Aim #2 studies will define the Ca2+-dependence of intra-acinar cell trypsinogen activation and the question of whether a rise in [CA2+]i is, by itself, sufficient to cause intra-acinar cell zymogen activation. The Specific Aim #3 studies are based on our recent finding that a phosphoinositide-3-kinase (PI3K) plays a central role in mediating zymogen activation. We will define the class of PI3K which is involved and elucidate the mechanism(s) by which it mediates zymogen activation. Finally, our Specific Aim #4 studies will build upon our recent findings which suggest that pancreatic acinar cells may possess proteinase-activated receptors (PARs) that regulate acinar cell expression of inflammatory mediators. We will determine if, in fact, acinar cells display PAR-2 and/or PAR-4 receptors, whether those receptors respond to other proteases in addition to trypsin, and whether PAR-mediated chemokine/cytokine expression is dependent upon changes in [CA2+]i homeostasis. Taken together, the studies proposed in this application will advance our understanding of the cellular basis for pancreatitis and of the events which regulate the severity of pancreatitis. They will identify potential targets for therapeutic intervention in this frequently devastating disease.